Cellular response to surface with nanoscale heterogeneous rigidity

ABSTRACT

An elastomeric substrate comprises a surface with regions of heterogeneous rigidity, wherein the regions are formed by exposing the elastomeric substrate to an energy source to form the regions such that the regions include a rigidity pattern comprising spots.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Application Ser. No. 61/895,068, filed on Oct. 24,2013, and is a continuation-in-part under 35 U.S.C. §120 of U.S. patentapplication Ser. No. 12/936,025, which is a U.S. National Stage Filingunder 35 U.S.C. §371 from International Application No.PCT/US2009/002069, filed Apr. 2, 2009 and published as WO 1009/123739 A1on Oct. 8, 2009, which claimed priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/072,717, filed on Apr. 2, 2008, eachof which are incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under PN2EY016586awarded by the National Institutes of Health (NIH) Common FundNanomedicine programme. The Government has certain rights in theinvention.

BACKGROUND

The physical properties of a cell's environment are important factors indetermining cell behavior and ultimately, phenotype. Among thesefactors, matrix rigidity can affect cell growth, differentiation andadhesion and motility. Alteration of the cellular rigidity sensingmechanism can be implicated in malignant transformation andtumerogenesis. Many aspects of the cellular rigidity-sensing mechanismare of interest, particularly in reference to tactile cell sensing ofdiscrete localized areas of increased or decreased rigidity.

As well as responding to biochemical signals, cells can directly probethe physical properties of the extracellular environment around them,such as to determine force, shape, geometry and stiffness of theextracellular environment. The cells can probe the extracellularenvironment by adhering and initiating matrix deformation by theapplication of cellular tractive forces. Matrix or tissue elasticity canhave a role in regulating multiple cell processes, including celladhesion, cell migration, and differential function, throughcell-generated actomyosin interactive forces regulated by a dynamicfeedback mechanism.

The sensitivity of cells to the mechanical properties of theextracellular matrix can be attributable to the mechanosensitive natureof the molecules involved in the structures of cell adhesion. Amongadhesive structures, focal adhesions appear to be the most prominent,demonstrating correlation between focal adhesion reinforcement andsustained force exhibiting a constant stress. This mechanosensitivitymay be regulated by a conserved local mechanism in which subcellularforces induce an elastic deformation of transmembrane integrin regions,triggering conformational and organizational changes and resulting inintegrin activation, which in turn can uncover cryptic binding sites foradditional protein binding, enabling focal adhesion reinforcement. Theseprocesses can set a dimensional scale for cellular rigidity sensing.

SUMMARY

The present disclosure describes elastomeric surfaces comprising regionsof heterogeneous rigidity at the micro- and nanoscale. The presentdisclosure also describes methods of making such surfaces, such as byexposing regions of an elastomeric film to an energy source, such as afocused electron beam (e-beam) or deep ultraviolet (UV) light, which canform patterned regions of micron or submicron spots, or both. Theexposed regions can undergo a non-linear increase in rigidity as afunction of the applied exposure (e.g., the electron dose or the UVdose). Cells cultured upon the surface can produce differentialfunctional responses based on heterogeneous rigidity patterning on thesurface, such as differential focal adhesion of the cells, differentialcell differentiation of the cells; differential immune response; ordifferential growth of the cells. For example, human skeletal stem cellscultured upon surfaces patterned in this manner displayed differentialfocal adhesion co-localization to the rigid regions, a behavior thatpersisted as the area of the exposed regions was reduced to ˜1 μm. Onspots with diameters of ˜250 nm and below, focal adhesionco-localization was lost. This implies that there exists a length scalefor cellular rigidity sensing, with the critical length in the range ofa few hundred nanometers.

In an example, the present disclosure describes an elastomeric substratecomprising a surface with regions of heterogeneous rigidity, wherein theregions are formed by exposing the elastomeric substrate to an energysource to form the regions such that the regions include a rigiditypattern comprising spots.

In another example, the present disclosure describes a method ofculturing cells, the method comprising culturing cells upon a surface ofan elastomeric substrate, the surface comprising regions ofheterogeneous rigidity, wherein the regions are formed by exposing theelastomeric substrate to an energy source to form the regions such thatthe regions include a rigidity pattern comprising spots.

In another example, the present disclosure describes a method forfabricating a substrate, the method comprising forming a substrate of anelastomer, the substrate having a surface, and exposing selected regionsof the surface to an energy source, the energy source configured tomodify a rigidity of the selected regions.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing/photographexecuted in color. Copies of this patent with colordrawing(s)/photograph(s) will be provided by the Office upon request andpayment of the necessary fee.

FIG. 1A shows a perspective view of a system for emitting an electronbeam onto a poly(dimethylsiloxane) (PDMS) thin-film for selectivemodification of a stiffness of the PDMS film.

FIGS. 1B and 1C show electron trajectory and scattering within a PDMSfilm, as determined by Monte Carlo simulations of exposure of PDMS toelectron beams.

FIG. 1D is a graph of the relationship between stiffness (e.g., modulus)as a function of electron beam exposure

FIG. 1E shows a graph of a function relating Young's modulus changes dueto the e-beam exposure as a function of E/E₀ where E is the modulus ofthe 3 μm PDMS film and E₀ is the modulus of a 120 μm spin-coated film.

FIGS. 2A and 2B show the physicochemical modulation of PDMS substratesby focused electron-beam patterning.

FIG. 2C shows a graph of the depth of pits in the elastomer as afunction of electron beam dose.

FIG. 2D shows a surface topography of a region of PDMS exposed to anelectron beam.

FIG. 2E shows a chart of the surface water contact angle of the PDMSboth before and after electron beam exposure as well as the contactangle for glass.

FIG. 2F shows an X-ray photoelectron spectroscopy analysis of a PDMSsurface both before and after electron beam exposure.

FIG. 2G shows a graph of the surface fibronectin distribution of PDMSafter electron beam exposure.

FIGS. 3A-3F show focal adhesion formation by mesenchymal stem cells on aPDMS surface having 2 μm spots with modulated rigidity, with FIG. 3Ashowing a mesenchymal stem cell on spots having the highest relativerigidity, and FIG. 3F showing a mesenchymal stem cell on spots havingthe lowest relative rigidity.

FIGS. 4 a-4 f show the same mesenchymal stem cells as shown in FIGS.3A-3F, respectively, but after paxillin staining of the mesenchymal stemcells, with FIG. 4 a showing the paxillin staining of the mesenchymalstem cell on spots having the highest relative rigidity, and FIG. 3 fshowing the paxillin staining on the mesenchymal stem cell on spotshaving the lowest relative rigidity.

FIG. 5 shows a graph of Mander's coefficient of colocalization as afunction of electron beam exposure dose, which in turn shows therelationship between colocalization and spot rigidity.

FIG. 6 shows a graph of the fluorescence intensity of focal adhesionsassociated paxillin as a function of electron beam exposure dose whichin turn shows the relationship between colocalization and spot rigidity.

FIGS. 7A-7F show focal adhesion formation by mesenchymal stem cells on aPDMS surface having spots with the same rigidity, but with modulatedspot diameter, with FIG. 7A showing a mesenchymal stem cell on spotshaving the largest relative spot diameter, and FIG. 7F showing amesenchymal stem cell on spots having the smallest relative spotdiameter.

FIGS. 8 a-8 f show the same mesenchymal stem cells as shown in FIGS.7A-7F, respectively, but after paxillin staining of the mesenchymal stemcells, with FIG. 8 a showing the paxillin staining of the mesenchymalstem cell on spots having the largest relative spot diameter, and FIG. 8f showing the paxillin staining on the mesenchymal stem cell on spotshaving the smallest relative spot diameter.

FIG. 9 shows a graph of Mander's coefficient of colocalization as afunction of spot diameter.

FIG. 10 shows a graph of the fluorescence intensity of focal adhesionsassociated paxillin as a function of spot diameter.

DETAILED DESCRIPTION

FIG. 1A shows a system 10 for selectively modifying a stiffness of anelastomer film 12 by exposing the elastomer film 12 to an energy source14, such as an electron beam 14, from an energy source 16, such as anelectron gun 16. In other examples, described in more detail below, theelastomer film 12 can be exposed to another energy source, such as deepultraviolet light (“deep UV”). The elastomer film 12 can be depositedonto a substrate 18, such as a glass substrate 18, which can providestructural support to the thin elastomer film 12.

The elastomer film 12 can be configured to provide a support for livingcell, wherein a heterogeneous rigidity pattern formed on an uppersurface 20 of the elastomer film 12 can be configured to affect acharacteristic of a living cell located on the upper surface 20 of theelastomer film 12. The heterogeneous rigidity pattern may be capable ofproducing a differential functional response in cells cultured on theelastomer based on heterogeneous rigidity patterning on the surface. Thedifferential response can include, but is not limited to, at least oneof differential focal adhesion of the cells, differential celldifferentiation of the cells (such as T-cell or stem celldifferentiation), differential immune response, or differential growthof the cells. For example, stem cells can show lineage-specificdifferentiation when cultured on substrates matching the stiffnesscorresponding to native tissue. Although tissues can be associated withYoung's modulus values for bulk rigidity, at the sub-cellular level, andparticularly at the micro- and nanoscales, tissues can be composed ofheterogeneous distributions of particles, and fibers of varyingrigidity. Skeletal stem cells can reside in a specialized biophysicaland biochemical niche environment within the medullary cavity. The typesof extra-cellular matrix architecture encountered by skeletal stem cellsin vivo can range from a fibrillar scaffold of connective tissue tonanophase apatite crystals of mineralization. A skeletal stem cell cantherefore encounter a composite environment of micro- and nanoscaletopographical features and discrete rigidities, with elastic moduliranging from 150 GPa (hydroxyapatite), to 5 GPa (collagen type I) or 2-7kPa (plasma membrane). How cells sense and respond to the mechanicalproperties of their surroundings in a heterogeneous rigidity environmentremains of interest. Other cells have been or are believed to respond toa heterogeneous rigidity pattern, such as the one formed on theelastomer film 12. Examples of cells that may respond to theheterogeneous pattern of the elastomer can include, but are not limitedto, stem cells, cancer cells, nerve cells (such as neurons), bone cells(such as osteoblasts), and muscle cells.

The elastomer film 12 can comprise a material that is capable of beingmodified by exposure to the energy source 14, for example by becomingmore rigid when exposed to the energy source 14. In an example, theelastomer film 12 can comprise poly(dimethylsiloxane) (PDMS), which isknown to undergo additional crosslinking when exposed to certain energysources 14, such as an electron beam 14 or deep UV. The elastomer film12 can, therefore, be modified to present cells with surfaces havingpatterns with specific rigidities approximating the range of rigiditiesencountered in physiological environments. For example, elastomers, andPDMS in particular, can be used in cell adhesion or migration assays, ormicrofluidic and MEMS technologies due to its favorable optical,biocompatible and mechanical properties. The PDMS film 12 can be used tostudy the role of extracellular rigidity on cellular adhesion anddifferentiation due to the ease by which its rigidity may be preciselycontrolled by simply varying the base:accelerator ratio.

PDMS substrates can be patterned into spots or other micron-scaledstructures, such as micron-scale pillars, that can facilitate themeasurement of focal adhesion reinforcement and cellular forces infibroblast cells. Because the rigidity can be dictated by a combinationof the elastomer composition and the structure dimensions, e.g. diameteror height, or both, the pliable deformation of dimensionally definedstructures by cellular forces can be precisely measured and used togenerate real-time force maps across entire cells. One recognizedshortcoming associated with elastomeric pillar structures is theintroduction of topographical features to the cell assay system, amodulator of focal adhesion formation and cellular function. In order tounderstand in more detail how cell behavior can be dictated by thearchitecture of the extra-cellular matrix rigidity, it can be helpful toisolate the mechanical basis of rigidity-mediated mechanotransductionfrom that of topographical sensing.

Patterned structure arrays, such as pillar or spot arrays, can be formedby a lithography-generated molding process. For example, PDMS can betransparent to visible and ultraviolet light and therefore cannot, ingeneral, be directly patterned by standard photolithography without theaddition of a photoactive compound. However, PDMS can be sensitive todeep ultraviolet light (deep UV) and electron beam (“e-beam”)irradiation, which can induce cross-linking of the elastomer. Thecross-linking, in turn, can alter surface mechanical properties of theradiation-exposed elastomer.

The present disclosure shows that exposure of an elastomer, such asPDMS, to radiation, such as e-beam or deep UV exposure, cansignificantly alter the rigidity of the elastomer. This allows for theformation of two-dimensional elastomeric substrates with geometricallypatterned heterogeneous rigidity. Exposed feature dimensions extendedfrom the macroscale to the micron scale and to the nanoscale and covereda range of rigidities, which can be regulated by varying the applieddose of the radiation, e.g., the e-beam exposure dose or the deep UVexposure dose. As described in more detail below, a differentialco-localization of focal adhesions to the patterned rigidity regionsoccurs with human skeletal stem cells, and this response can bemaintained on micron and submicron sized regions. Further, it has beenfound by the inventors that cells can integrate the pattern of rigidityto which they are exposed by regulating the localization of focaladhesions formed in order to maintain their spread shape. Thisadaptation can occur across a range of feature stiffness and size. Rigidspots as small as 250 nm across can induce the formation of localizedpunctate focal adhesions, but this effect is not observed on patternedregions having a size of 100 nm across. These observations reveal theexistence of a submicron machinery that may be used by cells to senselocal rigidity. Knowing these geometrical limits can yield a betterunderstanding of in vivo cell behavior in processes such asembryogenesis, wound healing and cellular metastasis. In addition, anunderstanding of the geometrical basis for rigidity sensing can behelpful for the design of implants with artificial smart surfacesallowing optimal interaction with cells in a tissue.

In an example, the elastomer film 12 can be a 120 μm-thick layer of PDMSthat is deposited onto a glass substrate 18, such as by spin-coatingPDMS onto the glass substrate 18. The glass substrate 18 can be treatedwith an oxygen plasma and coated with a final polymeric discharge layer22, such as AquaSAVE, prior to exposure of the elastomer film 12 to theenergy source 14.

The exposure of the energy source 14 onto the elastomer film 12 can becontrolled so that only certain portions of the upper surface 20 of theelastomer film 12 are exposed to the energy source 14. For example, ifthe energy source 14 is a focused electron beam 14, as shown in FIG. 1A,the focused electron beam 14 can be scanned over the substrate surface20 to create an array of defined spots 24. Alternatively, if the energysource 14 is a deep UV source 14, a mask (not shown) can be placed overthe upper surface 20 of the elastomer film 12 with openings through themask corresponding to the desired locations of the spots 24 in thearray, wherein the openings allow the deep UV light to pass through themask at the predetermined locations of the spots 24.

In an example, a PDMS mixture can be prepared at a 50:1 ratio of basePDMS polymer to accelerating agent. The PDMS mixture can be spin-coatedonto a substrate 18, such as a glass substrate 18. The PDMS mixture canbe cured for an extended period of time, such as at least about 1 hour,for example at least about 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours 11 hours,12 hours, 18 hours, or 24 hours, and at a temperature sufficient to curethe PDMS, such as at least about 50° C., for example at least about 55°C., at least about 60° C., at least about 65° C., or at least about 70°C., to form an optically transparent viscoelastic elastomer film 12. Theelastomer film 12 can be rendered hydrophilic by applying a short oxygenplasma treatment to facilitate a further spin-coating application of theelectrically conducting layer 22, also referred to as a discharge layer22, such as the polymer sold under the trade name AquaSAVE by MitsubishiRayon America, Inc., of New York, N.Y., USA, to suppress charging duringe-beam exposure.

In an example, the rigidity pattern of the elastomer film 12 can begenerated in the elastomer with e-beam exposure doses ranging from about500 microcoulombs per square centimeter (μC/cm²) to about 4,000 μC/cm².The electron beam 14 can be formed using an accelerating voltage ofabout 30 kilovolts (kV) and a beam current of about 2.5 nanoamperes(nA).

In the case of an electron beam 14, the absorbed electron energy withinthe PDMS film 12 can be determined by two factors: (i) the incidentelectron energy (such as about 30 kilo electron volts (keV)); and (ii)the scattering of electrons within the elastomer, which can depend onthe density of the elastomer. Monte Carlo simulation analysis wasconducted to determine the expected scattering of electrons in theelectron beam 14. The Monte Carlo simulation indicated that thepenetration depth exceeded about 15 micrometer (μm), but that over 90%of the e-beam energy was absorbed within approximately the top 3micrometer (μm) of the elastomer, as shown in FIG. 1B. As shown in FIG.1C, the penetration into PDMS can result in a scattering profile havinga columnar shape with a broad spreading base that diminishes inintensity with increasing depth, e.g., a general conical profile of theelastomer crosslinking Lateral scattering within the top layer was foundto be confined to about 30 nanometers (nm) at 30 keV.

Nanoindentation can be employed to characterize the change in theelastic properties of the elastomer as a function of exposure to theenergy source 14. In an example, measurements can be made on large areaexposures, such as at least about 1 square millimeter (mm²), forexposure doses up to about 6,000 μC/cm². In an example, a diamondflat-punch indenter tip of radius 76.4 μm was oscillated at a frequencyof 110 Hz as the nanoindenter head approached the sample. After contactwith the elastomer surface, the nanoindenter head can maintain aconstant load and deflect the springs 1.5 μm. The tip can remain incontact with the elastomer, oscillating about 500 nm, while recordingthe amplitude and phase of the force and displacement of the embeddedtip. As mentioned above, the Monte Carlo simulation indicated that mostof the electron energy is deposited in about a 3 μm thick layer at thesurface of the elastomer film 12. In order to extract the mechanicalproperties of this layer from the force-displacement curves, finiteelement analysis (FEA) can be applied to model a multilayer stackincluding the exposed layer, the elastomer film 12 and the underlyingsubstrate 18. The FEA calculations can also take into account theprestrain induced in the elastomer as a result of the spin coatingprocess and the contraction of the elastomer as a result of thecross-linking due to exposure to the energy source 14. The FEAcalculations can be determined from depth profiles of the exposedfeatures (as shown in FIGS. 2A-2D) and nanoindentation measurements onunexposed elastomer films (such as a 10 mm-thick PDMS block used as areference). The results of the nanoindentation and FEA curve-fitting, interms of the relative increase in Young's modulus for the top 3 μm ofthe elastomer film 12, are shown in FIG. 1D, indicating that exposure tothe energy source 14 can cause an increase in the stiffness of thepolymer, such as up to two orders of magnitude or more. FIG. 1D showsthat e-beam exposure of a PDMS film can cause an increase in stiffnessof the polymer due to crosslinking and also a reduction in volumeleading to increased pre-strain in the film. The increase in Young'smodulus can be bounded by the unexposed material properties and theexpected level of pre-strain due to e-beam exposure. FIG. 1D shows theresulting apparent μ_(storage) and tan(δ) as a function of e-beam dose.

The application of the energy source 14 to the elastomer can result inthe formation of shallow depressions surrounding the exposed regions.Without being bound to any theory, the inventors believe that this canbe attributed to minor contraction of the elastomer as a result of theexposure-induced cross-linking, which can cause circumferentialstressing beyond the periphery of the exposed region (see FIG. 2A andFIG. 2B). E-beam exposure doses of 4,000 μC/cm² initiate nanoscalecontractions of PDMS resulting in an undulating topography of shallowpits, as shown in FIGS. 2A and 2B. This topographical response can bedose dependent. As shown in FIG. 2C, low doses of 50 μC/cm² resulted inundulations of approximately 4 nm depth. PDMS contraction and pit depthwas observed to plateau with doses of 2,200 μC/cm², and resulting pitsmeasured about 140 nm in depth.

Although nanoscale pits have been found to reduce focal adhesionreinforcement when greater than 73 nm in depth, these effects can benegated when pit diameters are within the micron range and the featureradius of curvature is below about 200 nm (see FIG. 2D). FIG. 2D showsthe surface topography of PDMS after exposure to an electron beam, asdetermined by profilometry of the surface. Profilometry revealed thatdoses of up to 10,000 μC/cm² did not increase the pit depth. Theresultant topography was composed of smooth undulating pits with aradius of curvature of approximately 20 μm. In order to isolate thepossible involvement of the topographical modulation on cellularfunction, e.g., the topography arising from e-beam mediated elastomercontraction, on cellular function, control elastomer substrates werefabricated by imprinting with a negative elastomer shim to yieldtopographically yet non-rigidity modified elastomer substrates. Cellularresponse to these control substrates are described below in the Example.

Samples were cleared of aquaSAVE prior to physicomechanical analysis andthe chemical effects of e-beam irradiation on the elastomer wereassessed prior to in vitro cell experiments to ensure that themodulation of cellular function was exclusively rigidity dependent andnot as a result of altered surface hydrophobicity or protein adsorption,or both. According to oxygen plasma surface wettability analysis, thewettability of experimental elastomer substrates was analyzed by contactangle measurements (see FIG. 2E), which confirmed no significantdifferences in water contact angle between all the materials that formthe cellular support (e.g., PDMS and glass). X-ray photoelectronspectroscopy (XPS) analysis, however, indicated a significant reductionin the surface carbon profile of the elastomer when irradiated with anintermediate area dose. At a dose of about 1,000 μC/cm² the surfacecarbon content decreased by about 50% (see FIG. 2F).

In order to assess the protein and cellular response to heterogeneouslypatterned rigidities, elastomer substrates were patterned with an arrayof spots with diameters ranging from about 100 nm to about 2 μm and anedge-edge spacing of about 3 spot diameters and with the doses indicatedpreviously. The inter-spot distance was modulated proportionally withspot diameter in order to ensure that the cells were exposed to arelatively constant ratio of higher rigidity and lower rigidity.

The protein adsorption distribution can be assessed by analyzing thefluorescence intensity profile of labeled fibronectin vs. the brightfield differential interference contrast (DIC) intensity on the e-beamexposed materials and was analyzed with ImageJ. The bright fieldintensity increased sharply at the irradiated regions, demonstratingthat e-beam exposed elastomer can cause minimal lateral scatteringduring irradiation and indicating the presence of diffractivemodification. On the other hand, the fluorescence intensity profile wasunchanged at the sites of e-beam exposure, indicating a uniformdistribution of protein adsorption on the patterned substrates (FIG.2G).

Effect of Elastomer Rigidity on Focal Adhesion Formation

The effect of spot rigidity on the formation of focal adhesions wasinvestigated. Elastomer substrates were formed and exposed to e-beamradiation of varying doses in order to form spots having varyingrigidities (shown in FIGS. 3A-3F). The spots of each elastomer substrateshown in FIGS. 3A-3F had the same spot diameter of about 2 μm. Theelastomer substrates were sterilized in 70% ethanol and washed inphosphate-buffered saline solution (PBS) before seeding of immortalizedhuman mesenchymal stem cells (hMSCs) derived from human bone marrowaspirates and stably transduced by a retroviral vector containing thegene for the catalytic subunit of human telomerase (hTERT). The hMSCswere cultured on experimental elastomer substrates (e.g., substrateswhere the spots had been exposed to e-beam radiation) and controlsubstrates (e.g., unexposed substrates) for about 18 hours before fixingand preparing for immunocytochemistry. The hMSCs were stained for thefocal adhesion protein paxillin (FIGS. 4 a-4 f), and analysis of focaladhesion co-localization and intensity on the irradiated regions wasperformed with ImageJ image processing software.

Analysis of focal adhesion formation on exposed spot regions showed thatfocal adhesion co-localization to the exposed regions increased withapplied electron beam dose. On substrates patterned with 4,000 μC/cm²and spots measuring 2 μm in diameter, MSCs formed punctate focaladhesions that co-localized significantly with the underlying exposedregion. This effect was observed to diminish with decreasing spot dose(FIGS. 3A-3F, FIGS. 4 a-4 f, and FIG. 5). It has been found thatincreased focal adhesion colocalization appears to increase toapproximately 60% focal adhesion colocalization at a dose of at least4000 μC/cm². Cellular spreading or actin cytoskeleton organization doesnot appear to have affected MSCs cultured on 2 μm diameter spots ofmodulated rigidity (see FIGS. 3A-3F). However, paxillin staining (FIGS.4 a-4 f) revealed a significant increase in focal adhesioncolocalization and fluorescence intensity with increasing spotstiffness.

On spots having lower rigidity (e.g., less e-beam dosage), elongatedfocal adhesions were observed to initiate at the irradiated regions yetextended onto the unexposed inter-spot regions, also referred to as“soft” regions, and co-localization was eliminated with decreasingrigidity. Interestingly, fluorescence intensity of focal adhesionassociated paxillin was also significantly increased on stiff regions(FIG. 6) indicating increased recruitment of paxillin to focal adhesionsformed on the stiffer elastomer regions. Varying the spot rigidity wasalso observed to significantly modulate total focal adhesion area, butnot cellular spreading.

Effect of Rigid Area Size on Focal Adhesion Formation

The effect of the size of rigid areas of elastomer substrates was alsoinvestigated. Elastomer substrates were formed and exposed to e-beamradiation of the same dose, but the diameter of the spots that this dosewas applied to was varied resulting in rigid regions having varying spotdiameters (shown in FIGS. 7A-7F). The spots of each elastomer substrateshown in FIGS. 7A-7F were exposed to the same e-beam dose of 4000μC/cm². Analysis of focal adhesion co-localization to the rigid spots inthe substrates of FIGS. 7A-7F revealed that punctate focal adhesionco-localization was dependent on spot size. Reducing the exposed spotdiameter from 2 μm to 100 nm significantly decreased focal adhesionco-localization (FIGS. 7A-7F, FIGS. 8 a-8 f, and FIG. 9). Cellularspreading or actin cytoskeleton organization does not appear to haveaffected mesenchymal stem cells cultured on spots exposed to electronbeams having a dose of 4000 μC/cm², and with varying spot diameter (seeFIGS. 7A-7F). However, paxillin staining (FIGS. 8 a-8 f) revealed asignificant increase in focal adhesion colocalization and fluorescenceintensity with increasing spot diameter.

By decreasing the spot diameter and inter-spot spacing, colocalized,punctate focal adhesions became less frequent. Rather, focal adhesionswere observed to bridge between multiple spots. Interestingly, focaladhesions that bridged between multiple spots were associated withpunctate domains of increased paxillin fluorescence intensity which wereshown to occur at the irradiated regions (FIG. 10), indicating focaladhesion sensing machinery can initiate discrete protein reinforcementalong the focal adhesion plaque. Varying the spot diameter was observedto significantly modulate total focal adhesion area, yet not cellularspreading. However, varying the spot size was observed to affectcellular motility. Specifically, cell velocity and mean migrationdistance were significantly reduced on substrates patterned withdots >500 nm in diameter.

As noted above, deep ultraviolet light (“deep UV”) can also be used asthe energy source 14 that can trigger cross-linking within the elastomerfilm 12 in order to modify the stiffness of the elastomer. The term“deep ultraviolet light’ or “deep UV,” as used herein, can refer toultraviolet light having a wavelength of less than about 300 nanometersand greater than about 150 nanometers, such as from about 190 nanometersto about 260 nanometers. In some examples, UV light having a wavelengthof about 248 nanometers can be used, and in other examples UV lighthaving a wavelength of about 193 nanometers can be used. The deep UVlight can be patterned onto the elastomer film 12 using, for example, amask that allows passage of deep UV light onto some areas of theelastomer film 12, e.g., to form the array of spots 24, and blockspassage of the deep UV light from reaching other areas of the elastomerfilm 12, e.g., the areas between the spots 24.

In an example, the modification in rigidity of the elastomer film 12,e.g., the modified stiffness of the spots 24, can be achieved withoutrequiring different dimensions or sizes of structures at the uppersurface 20 of the elastomer film 12. Previously, micro- or nano-scalestructures, such as nano-scale pillars have been used to modulate therigidities of the substrate, where the size, shape, and/or dimensions ofthe pillars have been modified in different regions to provide fordifferent rigidities at the different regions. In contrast, theelastomer films 12 described herein and the methods described herein formaking them have been found to provide for different rigidities withinthe elastomer, e.g., PDMS, without having to modify micro- or nano-scalestructures, such as pillars. In other words, the methods describedherein can provide for modification of the rigidity of the materialitself, and not on the macro-level rigidity that can be provided to thematerial by physical characteristics of micro- or nano-scale structuresmade up of the material. The technique described herein (e.g., aselective increase of elastomer rigidity in a lithographically patternedfashion) can also be used on elastomeric surfaces that have been moldedinto three-dimensional structures such as pillars. The result would bepillared surfaces with locally variable stiffness.

Discussion

Integrin mediated adhesions play dual physiological functions—asphysical structures that direct and regulate tissue and organmorphogenesis, and as bi-directional mechanosensors that modulatecellular signaling events. Cells can be very sensitive to the physicalstate of the local environment. Matrix rigidity can be conveyed throughmacromolecular focal adhesions in a bidirectional manner, modulatingcellular function and focal adhesion morphology. Stiffer matrices cangenerate larger focal adhesions through increased intracellular tension,and study of bulk systems of rigidity indicates that cells respond torigidity gradients, and migrate from regions of lower to higherrigidity, in a process termed durotaxis.

Major differences in focal adhesion reinforcement and density areobserved in cells cultured on surfaces with bulk compliance (sub-kPa toa few kPa), relative to cells cultured on surfaces with bulk rigidity(hundreds of kPa to a few MPa). However, conflicting hypotheses exist onthe mechanisms of bulk-rigidity mediated changes to focal adhesionreinforcement and subsequent cell function. Cells can exert a mechanicalforce on individual fibers of the matrix and gauge the feedback to makecell-fate decisions. By increasing the density of binding sites within asystem, the number of individual sites available for focal adhesionformation is also increased. This contrasts with data indicating thataltered rigidity can induce changes to cellular function irrespective ofbinding site density.

In an example, direct write e-beam patterning can be used to producegeometrical patterns of increased rigidity on viscoelastic elastomerfilms 12, such as PDMS substrates. By controlling the size of spots inthe micrometer and submicrometer range, for example from about 2 μm toabout 100 nm, and an edge-to-edge distance (e.g., the distance betweenadjacent spots) of about 6 μm to about 300 nm, a substrata with defineddistributions of heterogeneously increased rigidity can be reliablycreated with dimensions ranging from the micro to the nanoscale.Furthermore, this observable increase in substrate rigidity was notassociated with modulations in binding site density or polymerconcentration.

To study the effects of discrete spatial rigidity on focal adhesionformation and cell morphology, the effects of substrate rigidity can beisolated from the influences of substrate topography and chemistry,which have been shown previously to modulate the reinforcement of focaladhesions, dynamic turnover of focal adhesion associated proteins, andintegrin mediated signaling activity.

As mentioned above, minor topographical modification, such as via thecontraction of the elastomer as a result of cross-linking induced byexposure to the energy source, such as an electron beam, was observedfor all doses. Shallow depressions were observed with increasingexposure. These features had depths which plateaued at 120 nm anddiameters which extended to a maximum of twice the original exposed dotdiameter. These depressions had a radius of curvature of approx. 20 μmand a feature height:radius of curvature ratio of approx. 1:200.Importantly, cellular studies into nanoscale feature curvature haverevealed a critical feature height:radius of curvature ratio of 1:1, andthat substrates possessing nanofeatures with radius of curvaturesgreater than this do not initiate contact guidance or modulate focaladhesion formation. In order to verify that the minor topographicalundulations produced by e-beam exposure indeed did not influence focaladhesion formation, we fabricated control “topography” substrates frominverse replica shims. Cells plated on control substrates displayed noresponse to the topography and no focal adhesion co-localization topatterned features as was seen on the e-beam exposed substrates (FIGS.3A-3F).

We note that, in contrast, some aspects if the PDMS surface chemistrycan be affected by focused e-beam exposure, such as a reduction in themeasured surface carbon content (FIG. 2F). This was also present inother observations that described a similar reduction in carbon contentin PDMS following exposure to UV light. Here the PDMS underwent asilica-like chemical transformation accompanied by an appropriate changein the bulk PDMS refractive index. Without wishing to be bound by anytheories, the inventors speculate that similar e-beam induced changes inPDMS refractive index, greatly aided in the bright field imaging of theexposed rigidity pattern, which, in fact, aided microscopy-basedco-localization analysis.

PDMS hydrophobicity can promote rapid non-specific protein adsorptiondue to inherent charges present along the protein molecule. Oxygenplasma was employed to enhance PDMS wettability and enable theapplication of a water-soluble electrical discharge layer to enablee-beam patterning. The plasma treatment can also aide in reducingsubstrate hydrophobicity as indicated by contact angle analysis andreported previously. As noted above (and shown in FIG. 2G), differentialfibronectin adsorption on heterogeneous rigidity substrates was notobserved, and it is therefore believed that the observed focal adhesionco-localization effects cannot be attributed to differences in proteinadsorption between the irradiated and non-irradiated regions.

Focal adhesion reinforcement and induction of actin organization requirecertain threshold densities of adhesion ligands. However, the existenceof a minimum length scale at which cells can sense rigidity is ofinterest. The results described herein demonstrate that the formation offocal adhesions in mesenchymal stem cells cultured on substrates withheterogeneous rigidity is dependent both on feature stiffness and size.The formation of discrete punctate focal adhesions coupled with anincrease in paxillin recruitment on spots ≧1 μm in diameter, indicatethat this length scale lies between 500 nm and 1 μm. Paxillinrecruitment to focal adhesions and subsequent phosphorolation can beessential for high focal adhesion traction over a broad range of ECMrigidities.

Additionally, a reduction in cell motility was observed when the cellswere cultured on stiff regions of increasing diameter. This observationdiffers significantly from durotaxis observations with bulk rigiditieswhere increased cellular motility is observed with increasing substratestiffness. Similarly, other observations showed significant increases incell motility when cultured on spots of clustered adhesion ligand. Thisseems to imply that the cellular response to adhesion ligand density andsubsequent and focal adhesion formation is mediated via independentmechanistic processes to those that govern focal adhesion reinforcementon regions of increased rigidity.

We also observed that on spots ≦1 μm in diameter, focal adhesions had atendency to extend paxillin domains, and single focal adhesions wereassociated with multiple rigid spots. The results described hereinsuggest that rigidity mediated adhesion can be regulated by the samemachinery that governs focal adhesion assembly and reinforcement andthat this machinery can be capable of recognizing localizeddiscrepancies in matrix rigidity.

Tissues do not represent bulk rigidity systems, but rather can becomposed of heterogeneous distributions of particles, and fibres ofvarying rigidity. We have developed a new type of biomimetic surfacecomprising regions of heterogeneous rigidity on the micro- and nanoscaleby writing on PDMS films with an electron beam. Finite element analysisof nanoindentation measurements on these surfaces reveal a substantialincrease in the Young's modulus of the elastomer as a result of thee-beam exposure. In a semi-planar system, the cellular rigidity sensingapparatus can be capable of sensing discrete submicron discrepancies inthe matrix rigidity, and that focal adhesions demonstrate intrinsic“local” reinforcement in response to rigid features measuring {tildeunder (>)}500 nm in diameter.

Different cell types can respond differently to rigidity and can havedifferent spatial and rigidity requirements to elicit particularbehaviors and responses. The versatility of the patterning systempresented here can be applied to a broad range of cellular systems inorder to elucidate the specific requirements for each. Understandingthese requirements can be used to guide the design of new types oftissue scaffolds and can have implications in the treatment of cancerand other diseases.

Example

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative example.

Substrate Fabrication

Microscope cover-glasses (Corning, NJ, USA) (22 mm2 No. 0) were cleanedfor 12 h in a 1% v/v solution of the detergent MICRO-90 (InternationalProducts, NJ, USA), rinsed in reverse osmosis water (ROH₂O) andblown-dry in a stream of filtered nitrogen. Sylgard 184 PDMS (DowCorning, MI, USA) was mixed with the supplied accelerating agent at aratio of 50:1 for 5 min and degassed under vacuum for 10 minutes at 5Torr. PDMS (0.5 ml) was applied to the microscope cover-glasses andspin-coated for 45 s at 1000 r.p.m. and an acceleration of 400 r.p.m.s⁻¹ to form a uniform film. The PDMS-coated cover-glasses were cured for17 h at 70° C. before further processing. Substrates were subjected toan oxygen plasma in a tabletop Harrick PDC32G plasma cleaner for 10seconds at a RF power of 18 W to induce surface hydrophilicity. Sampleswere next coated with a conductive discharge layer to facilitate e-beamexposure. A 5 nm thick discharge layer was applied to the substrates byspin coating 100 ul of AquaSAVE (Rayon, Mitsubishi) for 45 seconds at4000 rpm and an acceleration of 400 rpm. Samples were stored at roomtemperature until e-beam exposure.

Electron-Beam Direct-Write Patterning

The PDMS substrates were patterned by e-beam exposure using a scanningelectron microscope (FEI XL 30 Sirion) equipped with a Nabity NPGSpattern generator. A 1 mm² area consisting of an array of spots withdiameters ranging from 100 nm to 2 μM were written onto the substratesurface at doses from 500-4,000 μC/cm², an accelerating voltage of 30 kVand a beam current of approximately 2.5 nanoamperes. Substrates werecleared of AquaSAVE in deionized water three times for 5 minutes eachand allowed to air dry for 30 min.

Topographical control substrates were also fabricated by PDMS castingonto directly e-beam written samples to create negative shims. Briefly,e-beam written samples were coated with a silanized monolayer of 99.9%hexamethyldisilazane (Sigma-Aldrich) and the patterned area isolatedwith a glass cloning-ring. PDMS with a base:accelerator ratio of 5:1 wasintroduced into the cloning ring and allowed to cure overnight at roomtemperature. The inverse cloning-ring/PDMS shim was subsequently removedfrom the direct e-beam written pattern and coated with an anti-adhesivehexamethyldisilazane layer as above. Topographical PDMS substratesprepared as above were imprinted with the PDMS shim overnight to yieldtopographically yet non-rigidity modified PDMS substrates.

Cell Culture

Substrates were sterilized by successive rinsing in 70% ethanol 3 timesfor 5 seconds each, followed by phosphate-buffered saline solution (PBS)3 times for 5 seconds each. Immortalized human mesenchymal stem cells(hMSCs) derived from human bone marrow aspirates were stably transducedby a retroviral vector containing the gene for the catalytic subunit ofhuman telomerase (hTERT). Cells were expanded to passage following 5weeks of culture and subsequently trypsinized in TrypLE Expressdissociation medium (Invitrogen) and seeded onto untreated experimentaland planar control tissue culture plates at a density of 1×10⁴ cells persample in 1 mL of complete medium. Cells were maintained at 37° C. witha 5% CO₂ atmosphere in Dulbecco's modified Eagle's medium (Gibco)supplemented with 10% fetal bovine serum (Gibco), 1% 1-glutamine and 100IU mg⁻¹ penicillin/streptomycin (Invitrogen).

Fluorescent Labeling

Following 24 hours culture on experimental and control substrates, thehMSC cultures were fixed in 4% paraformaldehyde in phosphate-bufferedsaline solution (PBS), with 1% sucrose at 37° C. for 5 min. Once fixed,the samples were washed with PBS. Samples were permeabilized withbuffered 0.5% Triton X-100 (10.3 g sucrose, 0.292 g NaCl, 0.06 g MgCl₂,0.476 g [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (HEPES),0.5 mL Triton X-100, in 100 mL water, pH 7.2) at 4° C. for 5 min.Nonspecific binding sites were blocked with 1% bovine serum albumin(BSA) in PBS at 37° C. for 15 min and subsequently incubated for 2 hwith anti-paxillin monoclonal anti-human IgG raised in mouse, (1:200,(B.D Biosciences, Sparks, Md.). Nonspecific charges (e.g. remainingaldehyde) were neutralized with 0.5% Tween 20/PBS three times for 5 mineach to minimize background labeling. A secondary, Fluoresceinisothiocyanate-conjugated antibody was added, in 1% BSA/PBS, (1:50,Vector Laboratories, Burlingame, Calif.) at 4° C. for 1 h andsimultaneously, rhodamine-conjugated phalloidin was added for theduration of this incubation (1:50, Molecular Probes, OR). Substrateswere given a final wash in PBS 3 times for 5 min each Samples weremounted in Vectorshield mounting medium for fluorescence (VectorLaboratories, Burlingame, Calif.). Cell-substrate and cell-cellinteraction were examined by scanning confocal microscopy on a stagemaintained at 37° C. (live cell imaging). Imaging was performed on anLSM 700 scanning laser confocal microscope with an argon-ion laser(wavelengths 405; 488; 555; 639 nm) fitted with a Zeiss 100×α-PLANApochromat objective with a numerical aperture of 1.57 and with ZENsoftware (Carl Zeiss).

Time-Lapse Videomicroscopy

Time-lapse studies were performed. Briefly, MSCs were seeded ontopatterned and control PDMS substrata and incubated for 1 hour to allowcells to adhere. Cell media was subsequently removed and cells culturedin CO₂ independent medium (Gibeo) supplemented with 10% fetal bovineserum, 1% 1-glutamine and 100 IU mg⁻¹ penicillin/streptomycin(Invitrogen). The substrates were sandwiched to an aluminum microscopeslide with vacuum grease. Time-lapse micrographs were recorded with a20×, 0.7 NA air objective (Olympus) through a cooled CCD camera CoolSNAPHQ (Roper Scientific Inc.) using Simple PCI software (Compix Inc.).Images were captured via differential interference contrast (DIC)microscopy every 5 minutes.

Image Analysis

All images were analyzed using ImageJ (National Institutes of Health).Image stacks consisted of 2-3 planes spaced by 0.40 μm which wererendered using standard deviation image intensity to produce a singleimage of the ventral cell surface. Focal adhesions were analyzed incells from three separate experiments (20 cells each). Focal adhesionand exposed spot colocalization was analyzed by Manders' method with theJACoP plugin as described in Bolte et al., “A guided tour intosubcellular colocalization analysis in light microscopy,” J. Microsc,224, 213-232 (2006), incorporated herein by reference in its entirety.Manders' overlap coefficient is based on the Pearson's correlationcoefficient with average intensity values being taken out of themathematical expression, as described in Manders et al., “Dynamics ofthree-dimensional replication patterns during the S-phase, analysed bydouble labelling of DNA and confocal microscopy,” J. Cell Sci. 103 (Pt.3), 857-862 (1992). This coefficient will vary from 0 to 1, the formercorresponding to non-overlapping images and the latter reflecting 100%co-localization between both images. Therefore, M1 (or M2) determinedthe proportion of the fluorescent paxillin signal coincident with theDIC signal of the substrate over its total intensity, given as thefollowing: k₁=(Σ_(i)(A_(i, coloc)))/(Σ_(i) A_(i)) &k₂=(Σ_(i)(B_(i, coloc)))/(Σ_(i) B_(i)) With A_(i, coloc) being A_(i) ifB_(i)>0 and 0 if B_(i)=0; and B_(i, coloc) being B_(i) if A_(i)>0 and 0if A_(i)=0. Fluorescence intensity of focal adhesions was performed onpixels with a colocalization value of 1 relative to pixels with acolocalization value of 0. And plotted with the ImageJ plot profilefunction. Live-cell analysis of cell motility was performed with theImageJ plugin MTrackJ.

Surface Characterization

Monte Carlo simulations of electron trajectory in PDMS were conductedwith Casino software. Surface physical modification was characterized bynanoindentation, optical profilometry and scanning electron microscopymeasurements. Chemical modification was analyzed by water contact angle,angle, X-ray photoelectron spectroscopy (XPS) and confocal laserscanning microscopy measurements. Planar control materials were alsosubjected to a plasma treatment as described above in the sectionlabeled “Substrate Fabrication.”

Nanoindentation

An Agilent G200 nanoindenter Dynamic Contact Module (DCM) head was usedto investigate the dynamic modulus at 110 Hz. This frequency issufficiently close to the natural frequency of the DCM to benefit fromincreased sensitivity in the measurements. A diamond flat-punch indentertip of radius 76.4 μm was oscillated at 110 Hz as the nanoindenter headapproached the sample. Once contact with the surface of the PDMS wasdetermined, the DCM head maintained a constant load and deflects thesprings 1.5 μm. The tip remained in contact with the PDMS and oscillatedapproximately 500 nm, recording the amplitude and phase of the force anddisplacement of the embedded tip.

Surface Morphology

A Surface morphological assay was performed with a Veeco Wyko NT9100optical profiler with a 50× objective. Prior to imaging, samples weresputter-coated with a 12 nm layer of Au at 10 mA and 0.1 mbar using aCressington 108 sputter coater (Cressington, UK).

Electron Micrographs

Electron micrographs were obtained with a Hitachi S-4700 field-emissionscanning electron microscope (FESEM) fitted with an Autrata yttriumaluminum garnet (YAG) backscattered electron scintillator-type detector.The images were taken in secondary electron mode, with acceleratingvoltages between 2 and 5 kV. Images were taken with an emission currentof 20 μA, an aperture of 100 μm (apt1) and working distances of 10-12mm.

Contact Angle

Contact angle measurements were carried out at room temperature using 8μl water droplets with a model 100_(—)00 contact angle goniometer(Rame-Hart, Inc.). Values were averages of measurements on more thanthree different samples at more than three different locations on eachsample.

X-Ray Spectroscopy

X-ray photoelectron spectroscopy (XPS) spectra were recorded with PHI5500 model spectrometer equipped with a Al K monochromator X-ray sourcerun at 15 kV and 23.3 mA, a hemispherical electron energy analyzer and amultichannel detector. The test chamber pressure was maintained below2×10⁻⁹ Torr during the spectrum acquisition. Low energy electron floodgun was used to neutralize possible surface charging. The XPS bindingenergy (BE) was internally referenced to aliphatic main C 1 s peak(BE=284.6 eV). Survey spectrum was acquired at an analyzer pass energyof 93.9 eV and BE resolution of 0.8 eV, while the high-resolutionspectrum was acquired with a pass energy of 23.5 eV and BE resolution0.05 eV. Angle-dependent XPS was performed by rotating the sample holderto the desired take-off angle (the angle between the surface normal andthe detector) through a motor. Spectrum was fitted by a Guassian-Lorentz(BE) was internally referenced to aliphatic main C 1 s peak functionafter subtracting a striped background using the PHI data processingsoftware package under the constraint of setting reasonable BE shift andcharacteristic full width at high maximum (FWHM) range. Atomicconcentration was calculated by normalization of the peak area to theelemental sensitivity factor data provided by PHI database.

Fibronectin Adsorption

Surface adsorption of fibronectin was analyzed by fluorescencemicroscopy. Human fibronectin (Sigma Aldrich) was conjugated directly toAlexa Fluor 488 (Invitrogen) by protein dialysis according tomanufacturer's instructions (Thermo Scientific). Exposed substrates wereprepared as above and immersed in PBS containing 0.5 μg/ml fluorescentfibronectin. Samples were coated for 18 hours before being washed in PBS3 times for 5 minutes each and mounted for microscopy.

Statistical Analysis

All statistical analysis were performed with SPSS Statistics software 20(IBM, USA). Data are expressed as mean±SEM with * and ** indicating a95% and 99.5% confidence interval respectively. ANOVA was used todetermine statistical significance followed by post hoc Bonferoni'smultiple test correction to determine which groups were statisticallydifferent.

The above Detailed Description is intended to be illustrative, and notrestrictive. For example, the above-described examples (or one or moreelements thereof) can be used in combination with each other. Otherembodiments can be used, such as by one of ordinary skill in the artupon reviewing the above description. Also, various features or elementscan be grouped together to streamline the disclosure. This should not beinterpreted as intending that an unclaimed disclosed feature isessential to any claim. Rather, inventive subject matter can lie in lessthan all features of a particular disclosed embodiment. Thus, thefollowing claims are hereby incorporated into the Detailed Description,with each claim standing on its own as a separate embodiment. The scopeof the invention should be determined with reference to the appendedclaims, along with the full scope of equivalents to which such claimsare entitled.

In the event of inconsistent usages between this document and anydocuments so incorporated by reference, the usage in this documentcontrols.

In this document, the terms “a” or “an” are used, as is common in patentdocuments, to include one or more than one, independent of any otherinstances or usages of “at least one” or “one or more.” In thisdocument, the term “or” is used to refer to a nonexclusive or, such that“A or B” includes “A but not B,” “B but not A,” and “A and B,” unlessotherwise indicated. In this document, the terms “including” and “inwhich” are used as the plain-English equivalents of the respective terms“comprising” and “wherein.” Also, in the following claims, the terms“including” and “comprising” are open-ended, that is, a molding system,device, article, composition, formulation, or process that includeselements in addition to those listed after such a term in a claim arestill deemed to fall within the scope of that claim. Moreover, in thefollowing claims, the terms “first,” “second,” and “third,” etc. areused merely as labels, and are not intended to impose numericalrequirements on their objects.

Method examples described herein can be machine or computer-implemented,at least in part. Some examples can include a computer-readable mediumor machine-readable medium encoded with instructions operable toconfigure an electronic device to perform methods or method steps asdescribed in the above examples. An implementation of such methods ormethod steps can include code, such as microcode, assembly languagecode, a higher-level language code, or the like. Such code can includecomputer readable instructions for performing various methods. The codemay form portions of computer program products. Further, in an example,the code can be tangibly stored on one or more volatile, non-transitory,or non-volatile tangible computer-readable media, such as duringexecution or at other times. Examples of these tangiblecomputer-readable media can include, but are not limited to, hard disks,removable magnetic disks, removable optical disks (e.g., compact disksand digital video disks), magnetic cassettes, memory cards or sticks,random access memories (RAMs), read only memories (ROMs), and the like.

The Abstract is provided to comply with 37 C.F.R. §1.72(b), to allow thereader to quickly ascertain the nature of the technical disclosure. Itis submitted with the understanding that it will not be used tointerpret or limit the scope or meaning of the claims.

Although the invention has been described with reference to exemplaryembodiments, workers skilled in the art will recognize that changes maybe made in form and detail without departing from the spirit and scopeof the invention.

What is claimed is:
 1. An elastomeric substrate comprising a surfacewith regions of heterogeneous rigidity, wherein the regions are formedby exposing the elastomeric substrate to an energy source to form theregions such that the regions include a rigidity pattern comprisingspots.
 2. The elastomeric substrate of claim 1, wherein the regionsprovide for a differential functional response from cells cultured uponthe rigidity pattern of the regions.
 3. The elastomeric substrate ofclaim 2, wherein the differential functional response comprises at leastone of: differential focal adhesion of the cells; differential celldifferentiation of the cells; differential immune response; or growth ofthe cells.
 4. The elastomeric substrate of claim 2, wherein the cellscomprise at least one of stem cells, T-cells, cancer cells, nerve cells,osteoblasts, and muscle cells.
 5. The elastomeric substrate of claim 2,wherein at least some of the spots include a lateral dimension that isgreater than or equal to 250 nanometers.
 6. The elastomeric substrate ofclaim 2, wherein at least some of the spots include a lateral dimensionthat is less than or equal to 250 nanometers.
 7. The elastomericsubstrate of claim 1, wherein the energy source comprises at least oneof a focused electron beam or deep ultraviolet light.
 8. The elastomericsubstrate of claim 1, wherein the elastomeric substrate comprisespoly(dimethylsiloxane) or a poly(dimethylsiloxane)-based polymer.
 9. Theelastomeric substrate of claim 1, wherein the regions of heterogeneousrigidity are formed at at least one of a microscale or a nanoscale sothat the spots comprise micrometer or submicrometer scale spots.
 10. Amethod of culturing cells, the method comprising culturing cells upon asurface of an elastomeric substrate, the surface comprising regions ofheterogeneous rigidity, wherein the regions are formed by exposing theelastomeric substrate to an energy source to form the regions such thatthe regions include a rigidity pattern comprising spots.
 11. The methodof claim 10, wherein the regions provide for differential functionalresponse from cells cultured upon the rigidity pattern of the regions.12. The method of claim 11, wherein the differential functional responsecomprises at least one of: differential focal adhesion of the cells;differential cell differentiation of the cells; differential immuneresponse; or differential growth of the cells.
 13. The method of claim10, wherein the energy source comprises at least one of a focusedelectron beam or deep ultraviolet light.
 14. The method of claim 10,wherein the elastomeric substrate comprises poly(dimethylsiloxane) or apoly(dimethylsiloxane)-based polymer.
 15. The method of claim 10,wherein the regions of heterogeneous rigidity are formed at at least oneof a microscale or a nanoscale so that the spots comprise micrometer orsubmicrometer scale spots.
 16. A method for fabricating a substrate, themethod comprising: forming a substrate of an elastomer, the substratehaving a surface; and exposing selected regions of the surface to anenergy source, the energy source configured to modify a rigidity of theselected regions.
 17. The method of claim 16, wherein the energy sourcecomprises at least one of a focused electron beam or deep ultravioletlight.
 18. The method of claim 16, wherein the energy source initiatescross-inking of the elastomer in order to increase rigidity of theelastomer in the selected regions.
 19. The method of claim 16, whereinthe elastomer comprises poly(dimethylsiloxane) or apoly(dimethylsiloxane)-based polymer.
 20. The method of claim 16,wherein the selected regions are at at least one of a microscale or ananoscale.
 21. The method of claim 16, wherein forming the substratecomprises forming the surface to have three-dimensional structureswithin the selected regions of the surface, and wherein exposing theselected regions to the energy source comprises locally modifying arigidity of the three-dimensional structures.